title: Polysialic acid on neuropilin-2 is exclusively synthesized by the polysialyltransferase ST8SiaIV and attached to mucin-type o-glycans located between the b2 and c domain.
abstract: Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.
authors.affiliation: Institute of Cellular Chemistry, Medical School Hannover, Hannover 30623, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Rollenhagen
authors.forename: Falk F R
authors.initials: FF
authors.lastname: Buettner
authors.forename: Marc
authors.initials: M
authors.lastname: Reismann
authors.forename: Adan Chari
authors.initials: AC
authors.lastname: Jirmo
authors.forename: Melanie
authors.initials: M
authors.lastname: Grove
authors.forename: Georg M N
authors.initials: GM
authors.lastname: Behrens
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
authors.forename: Martina
authors.initials: M
authors.lastname: Mühlenhoff
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D039943:Neuropilin-2; D012794:Sialic Acids; C581308:neuropilin-2, human; C021319:polysialic acid; D012799:Sialyltransferases; C525848:ST8SiaIV protein, mouse; C515117:ST8SIA4 protein, human
keywords: Dendritic Cells; Glycoprotein; Glycosylation; Glycosyltransferases; O-Glycan Analysis; Polysialic Acid; Post-translational Modification